Nucleic Acids, i.e. DNA and RNA which are naturally occurring biopolymers, have been recognized for their ability to form different structures and conformations which might be exploited to create complex superstructures in two and three dimensions using these polymers (Amato, 1993--See list of references.). Such "nanostructures" could find use in different applications, particularly as scaffolds for the attachments of other important molecules, such as enzymes, for use in bioreactors.
The major approach towards making these nanostructures has made use of stable multi-way, three-way, four-way, or higher, DNA double-helical junctions (Chen and Seeman, 1991; Seeman, 1991; Seeman et al., 1994). The approach in this line of work is to generate stable four-way junctions, a DNA structure in which four double-helical elements are joined together to give a cross-shaped or a scissor-shaped structure with four double-helical "arms". To form these structures, four synthetic DNA single-stranded molecules are assembled together with the appropriately complementary sequences. The assembled four-way junction is designed also to have "sticky" ends--approximately six unpaired nucleotides at the terminus of each of its four double-helical elements, which are able to transiently bind other, complementary "sticky" ends from other four-way junctions or double-helices. These transient bindings, mediated by Watson-Crick base-pairing, are then rendered permanent and stable by ligation with enzymes such as T4 DNA ligase.
Using this general strategy, complex geometric objects, with the topology--if not the shape--of cubes and octahedra have been constructed out of double-helical DNA (Chen and Seeman, 1991; Seeman, 1991; Seeman et al., 1994). The strategy for these constructions, however, has multiple steps, involving repeated events of prior assembling of the component four-way junctions, with their collection of sticky ends. The interaction of sticky ends, involving Watson-Crick interactions between single-stranded complementary elements, are difficult to control due to their rapid occurrence in aqueous conditions in the presence of almost any kind of salt and the need to ligate the sticky ended interactions, which requires the use of expensive ligating enzymes and accessory chemicals such as adenosine triphosphate (ATP). The efficiency of the enzymatic ligation reaction is all-important for the overall success of the enterprise--a lack of complete ligation in a multiple-step assembly procedure can severely compromise the overall yield of the final, desired product.
Another class of DNA-based superstructures has attempted to utilize the formation of guanine-guanine quartets to hold together component single-stranded DNA molecules in extended quadruple helices (G4-DNA) (Sen and Gilbert, 1991). Only single-stranded DNA molecules, containing guanine-rich motifs, are capable of forming these structures, summarized under the general description of "G-wires" (Lu et al., 1992; Sen and Gilbert, 1992; Marsh and Henderson, 1994; Chen, 1995; Marsh et al., 1995). A very important disadvantage to the G-wires technology is that the formation of these wires is extremely difficult to control. Products are invariably polydisperse, containing a variety of sizes and molecular weight classes. The precise nature of bonding is also difficult to control, as is the difficulty of creating branched structures. With the current state of know-how, it would be very difficult if not impossible to create precise geometric shapes, such as cubes, or even extended networked arrays of DNA sequences using the G-wire technology
It is one of the objects of the present invention to obviate or litigate the above disadvantages with a new method for synapsing nucleic acids to create a nucleic acid superstructure. The term `synapsis` is used to denote the ability of one nucleic acid to bind another nucleic acid at specific, predetermined sites.
One of the applications for which a nucleic acid superstructure may be used is signal amplification. Many of the high-sensitivity detection systems that are widely used in bio-medicine nowadays are based on signal amplification technologies. Some commonly used techniques are radioimmunoassays (RIA), ELISA methods, as well as Western Blots. These techniques detect very low levels of a specific protein, nucleic acid, or other antigens in biological samples such as bodily fluids like blood, semen, and saliva, or in environmentally important samples such as water from rivers and other bodies of water. These techniques also provide the foundation for diagnostic assays used widely to monitor diseases such as AIDS. Most signal amplification technologies use antibodies, in which specific protein (antibody) molecules are used both to recognize and bind specifically to the antigen molecule in question and then to amplify the `signal` of the initial recognition event between antibody and antigen by many orders of magnitude. Without this amplfication step, the initial recognition event would be of low sensitivity, and therefore of little practical use. Thus, it is the amplification procedures which determine the sensitivity and, to a degree, the specificity of the diagnosis from the RIA, ELISA, and Western Blot techniques.
Fundamentally, in the techniques mentioned above, the initial recognition and binding of typically an antibody to an antigen molecule is followed by the binding of another set of antibodies to the first antibody. Thus, a monoclonal mouse anti-HIV antibody molecule might bind to an immobilized HIV antigen molecule in an ELISA plate. This is followed by the binding of secondary antibodies, eg. goat anti-mouse antibodies to the first, mouse antibody. Tertiary antibodies, eg. sheep anti-goat antibodies, are then bound to the secondary goat antibodies, and so on. At each stage of such secondary, tertiary, and further antibody additions, there is an amplification of the `signal`; in other words, two goat antibody molecules bind to each mouse antibody, and two sheep antibodies bind to each goat antibody, and so on. In this way, a `cascade` of antibody binding is produced. The end result is that numerous final antibodies may be immobilized to a single antigen molecule. The final antibodies may have a reporter molecule that may be used to produce a detectable signal of some sort eg. radioactivity, fluorescent labelling, or the activity of a coupled enzyme such as alkaline phosphatase.
It is, thus, another object of the invention to provide a novel and alternative means to antibodies as a basis for a signal amplification system by using synapsable DNA superstructures.
A number of investigators Murphy et al., 1993; Arkin et al., 1996! have demonstrated the fast and relatively easy passage of electrons through a double helix. The delocalized pi-electrons of the stacked base-pairs at the heart of the double-helix are presumably the path of electron transfer. Murphy et al. have studied this system by intercalating an electron-donating inorganic metal complex between base pairs at one end of a short double helix, and by positioning an electron-accepting metal complex at the other end of the double-helix.
Overall, these experiments have raised the interesting possibility of using DNA as a wire in nano-technological applications. It is conceivable that simple electronic devices, such as inductors, capacitors, diodes, and transistors, might be designed from DNA. The size range of DNA-in the nanometer to micrometer size domain, its electron-conducting ability, its precise structural dimensions, and its ease of automated synthesis make it eminently suitable for the above goal.
However, there are some special problems associated with the use of DNA as a material for designing circuits. An important issue, and one that has not been discussed in the literature, is the problem of making conducting junctions with double-helical DNA. A three-way double-helical junction is shown in FIG. 1a and FIG. 1b and a four-way double-helical junction is shown in FIG. 2a. The conduction of electricity through double-helical structures in general is contingent on a continuous stacking of base-pairs. However, the folded forms of three-way and four-way junctions are such that one or more helical elements are invariably not stacked on the others, out of purely geometric constraints. The three-way junction, for instance, can exist in either as a `Y` structure, shown in FIG. 1a, or as a `T` structure, shown in FIG. 1b, (Duckett & Lilley, 1990). Which structure is adopted is dependent on the identity of the base-pairs at the junctions. It can be seen that in either conformation, these conventional three-way junctions do not allow passage of electrons from an input arm to all of the other arms.
In a standard four-way junction made up of four double-helical elements, as shown in FIG. 2a, the double-helices arrange themselves into two sets each of two stacking double-helices; however, between each set there is no stacking of base pairs. Consequently an electron entering through any one of the double helices would be propagated rapidly through only its stacking partner, and not to the other two double-helical elements. Thus, a standard four-way junction also does not allow passage of electrons from an input arm to all of the other arms.
It is, therefore, another object of the invention to provide a better means for constructing conducting junctions using synapsable DNA superstructures.
It is yet another object of the invention to provide other applications of synapsable DNA superstructures.